EP2247728A1 - Sélection d'aptamères d'arn comme agents anti-malaria - Google Patents

Sélection d'aptamères d'arn comme agents anti-malaria

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Publication number
EP2247728A1
EP2247728A1 EP09708523A EP09708523A EP2247728A1 EP 2247728 A1 EP2247728 A1 EP 2247728A1 EP 09708523 A EP09708523 A EP 09708523A EP 09708523 A EP09708523 A EP 09708523A EP 2247728 A1 EP2247728 A1 EP 2247728A1
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seq
aptamer
rna
ggu
aptamers
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EP2247728B1 (fr
EP2247728A4 (fr
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John Lindh
Tina Persson
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APTAHEM AB
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Forskarpatent I SYD AB
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/16—Aptamers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/353—Nature of the modification linked to the nucleic acid via an atom other than carbon
    • C12N2310/3533—Halogen
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to particular RNA-aptamers against a conserved region of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMPI) to be used as an anti- malaria agent. Further, the invention relates to the use of such aptamers for the diagnosis of severe contra less severe malaria.
  • PfEMPI Plasmodium falciparum erythrocyte membrane protein 1
  • Aptamers are nucleic acid molecules having specific binding affinity to molecules through interactions other than classic Watson-Crick base pairing.
  • Aptamers like peptides generated by phage display or monoclonal antibodies (“mAbs"), are capable of specifically binding to selected targets and modulating the target's activity or binding interactions, e.g., through binding aptamers may block their target's ability to function. Discovered by an in vitro selection process from pools of random sequence oligonucleotides, aptamers have been generated for over 130 proteins including growth factors, transcription factors, enzymes, immunoglobulins, and receptors.
  • mAbs monoclonal antibodies
  • a typical aptamer is 10-15 kDa in size (20-45 nucleotides), binds its target with nanomolar to sub-nanomolar affinity, and discriminates against closely related targets (e.g., aptamers will typically not bind other proteins from the same gene family).
  • a series of structural studies have shown that aptamers are capable of using the same types of binding interactions (e.g., hydrogen bonding, electrostatic complementarities, hydrophobic contacts, steric exclusion) that drive affinity and specificity in antibody-antigen complexes.
  • Aptamers have a number of desirable characteristics for use as therapeutics and diagnostics including high specificity and affinity, biological efficacy, and excellent pharmacokinetic properties, in addition, they offer specific competitive advantages over antibodies and other protein biology, for example:
  • aptamers can be administered by subcutaneous injection (aptamer bioavailability via subcutaneous administration is >80% in monkey studies (Tucker et ah, J. Chromatography B. 732: 203- 212, 1999)). With good solubility (>150 mg/mL) and comparatively low molecular weight (aptamer: 10-50 kDa; antibody: 150 kDa), a weekly dose of aptamer may be delivered by injection in a volume of less than 0.5 ml_.
  • the small size of aptamers allows them to penetrate into areas of conformational constrictions that do not allow for antibodies or antibody fragments to penetrate, presenting yet another advantage of aptamer-based therapeutics or prophylaxis.
  • aptamers are chemically synthesized and consequently can be readily scaled as needed to meet production demand. Whereas difficulties in scaling production are currently limiting the availability of some biologies and the capital cost of a large-scale protein production plant is enormous, a single large-scale oligonucleotide synthesizer can produce upwards of 100 kg/year and requires a relatively modest initial investment.
  • the current cost of goods for aptamer synthesis at the kilogram scale is estimated at $500/g, comparable to that for highly optimized antibodies. Continuing improvements in process development are expected to lower the cost of goods to ⁇ $100/g in five years.
  • aptamers are chemically robust. They are intrinsically adapted to regain activity following exposure to factors such as heat and denaturants and can be stored for extended periods (>1 yr) at room temperature as lyophilized powders.
  • the aptamer discovery process readily permits lead modification, such as aptamer sequence optimization and the minimization of aptamer length [Conrad et al. 1996, Eaton et al. 1997].
  • 2' modifications such as 2 -fluoro and 2'-0-Me may be utilized for stabilization against nucleases without compromising the aptamer binding interaction with the target. See e.g. Lin et a. Nucleic Acids Res.
  • Severe malaria is almost exclusively caused by P. falciparum infection and usually arises 6-14 days after infection. Consequences of severe malaria include coma and death if untreated — young children and pregnant women are especially vulnerable. Splenomegaly (enlarged spleen), severe headache, cerebral ischemia, hepatomegaly (enlarged liver), hypoglycemia, and hemoglobinuria with renal failure may occur. Renal failure may cause blackwater fever, where hemoglobin from lysed red blood cells leaks into the urine. Severe malaria can progress extremely rapidly and cause death within hours or days. In the most severe cases of the disease fatality rates can exceed 20%, even with intensive care and treatment. In endemic areas, treatment is often less satisfactory and the overall fatality rate for all cases of malaria can be as high as one in ten. Over the longer term, developmental impairments have been documented in children who have suffered episodes of severe malaria.
  • Plasmodium falciparum is the causative agent of severe malaria in humans. Millions of people worldwide are infected every year by P. falciparum and more than one million die, most of them small children in sub-Saharan Africa.
  • the choice of drugs for malaria treatment has in the past primarily been quinine and chloroquine and since the 1960s sulfadoxine/pyrimethamine (SP).
  • SP sulfadoxine/pyrimethamine
  • parasite resistance towards all these drugs has been documented in endemic regions.
  • the most efficient drug used today as a first-line treatment is artemisinin and its derivates. Although no clinical resistance towards artemisinin has been demonstrated, there are indications of a developed in vitro resistance towards the drug in P. falciparum. Non-sterile immunity against severe malaria in residents in endemic regions has been described. This indicates the existence of antigenic homogeneity in the parasites causing severe malaria.
  • the protein known to be responsible for severe cerebral malaria by the process of rosetting and endothelial adherence is the erythrocyte membrane protein 1 (PfEMPI) expressed on the surface of the infected erythrocyte. Exposed on the infected erythrocyte it binds to a number of human cell surface receptors such as heparin sulphate, ICAM-1 , CD36, CSA, enabling the parasite to adhere to the endothelial linings of small blood vessels (cytoadherence) as well as to non-infected erythrocytes (rosetting), thus preventing spleenic clearance from the bloodstream. Such sequestered parasites cause considerable obstruction to tissue perfusion.
  • PfEMPI consist mainly of duffy binding ligand domains (DBL's) and cysteine rich inter domain regions (ClDR's), and the number of domains and size of the protein varies depending on which of the 60 var- genes is expressed.
  • DBL's duffy binding ligand domains
  • ClDR's cysteine rich inter domain regions
  • Certain sequence conservation is though expected due to the adhesive function of this protein.
  • the virulence-associated phenotype, rosetting, is mediated by the N-terminal Duffy-binding-like domain (DBLIalfa) which has a high degree of sequence conservation among the PfEMPI domains.
  • DBLIalfa N-terminal Duffy-binding-like domain
  • DBL1 ⁇ The definitive structure of DBL1 ⁇ is not known though extensive modelling on domains with similar structure has been performed. This makes DBL1 an attractive candidate for the development of novel drugs against severe malaria. Attempts of antibody recognition of the structural conserved epitopes in DBL1 ⁇ have been made without greater success due to the fact that the conserved regions are somewhat masked by the variable ones making them inaccessible to the comparatively large antibody.
  • RNA aptamers designed a Systematic Evolution of Ligand by Exponential Enrichment (SELEX) protocol, which could be designed to allow the selection of RNA aptamers to bind with high affinity and specificity to the structurally conserved parts of DBL1 ⁇ .
  • SELEX Systematic Evolution of Ligand by Exponential Enrichment
  • RNA aptamers that bind to cell adhesion receptors Trypanosoma cruzi and inhibit cell invasion.
  • a similar strategy has been reported for other pathogenic parasites where aptamers have successfully been selected against virulent surface proteins.
  • high affinity RNA aptamers were selected against the variable surface glycoprotein (VSG) of Trypanosoma brucei that proved to be capable of directing antibodies to the surface of live trypanosomes.
  • VSG variable surface glycoprotein
  • the other reported study used a different approach. The selection was not performed using expressed surface proteins from Trypanosoma cruzi but using a displacement technique with 4 different human surface receptors (Ulrich , H. et al, supra).
  • Trypanosoma cruzi causes heart problems in the chronic stage, whereby treatment involves managing the clinical manifestations of the disease. For example, pacemakers and medications for irregular heartbeats may be life saving for some patients with chronic cardiac disease, while surgery may be required for megaintestine. The disease cannot be cured in this phase, however.
  • Chronic heart disease caused by Chagas disease is now a common reason for heart transplantation surgery. Until recently, however, Chagas disease was considered a contraindication for the procedure, since the heart damage could recur as the parasite was expected to seize the opportunity provided by the immunosuppression that follows surgery.
  • aptamers have been shown to present the same high specificity and affinity for their targets as antibodies. In addition to efficient binding, aptamers also display an inhibitory activity of their targets.
  • the SELEX method is based on an iterative process of repeating steps of in vitro selection cycles where the initial DNA/RNA library of 10 14 - 10 15 different molecules is reduced to a smaller pool of approximately 100 different molecules that have high affinity towards the target in question.
  • Hjalmarsson, K. et al, FOI-R-1216-SE discusses Aptamers - Future tools for diagnostics and therapy, and is in particular related to the SELEX-methodoly. An overview of different aptamers and their optional use is given.
  • G ⁇ ringer, H. U., et al, Int. J. Parasit. 33 (2003), 1309-1317 discloses in vitro selection of high-affinity nucleic acid ligands to parasite target molecules, and discusses malaria, whereby the authors concluded primarily that the process of development has been slow because of the random screening methods of components have not been successful at identifying anti-parasitic compounds. The authors concentrate of discussing the SELEX protocol. Although malaria is a first parasite to be mentioned there is no hint that it adverse effects can be treated using an aptamer, but the authors discuss Trypanosoma bruzei causing sleeping sickness.
  • WO 2004/080420 to Mota et al discloses in general terms a methods for preventing or inhibiting the activity of malaria in vivo by administering an antimalarial agent to a mammal in need thereof, and indicates a number of different pathways to become interfered.
  • the disclosure discusses MET inhibition, MET being the recerptor for Hepatocyte Growth Factor.
  • the disclosure even mentions aptamers without any specification.
  • the disclosure is very conceptual without delivering any specific solutions, but only thoughts about possible routes for treating malaria.
  • the present inventors further show that these specific high affinity binding aptamers are able to bind to PfEMPI on the surface of live parasite and, lastly, that they have the capacity to disrupt rosettes.
  • specific binding of a set of aptamers that have an effect in vitro in formation of rosettes. It is further proposed to use these aptamers to diagnose the presence or not of severe malaria, i.e., to discriminate between light (or rather less severe forms) and severe forms of malaria.
  • the present invention relates to certain RNA-aptamers or active fragments thereof raised against PfEMPI as an anti-malarian agent.
  • the present invention relates to certain RNA-aptamers raised against the semi- conserved duffy binding ligand domain 1 ⁇ , DBL1 ⁇ , region of the Plasmodium falciparum erythrocyte membrane protein 1 , PfEMPl
  • UAAUUAUCCCG SEQ. ID. NO. 6
  • active fragment thereof SEQ. ID. NO. 6
  • the invention relates to an aptamer having the sequence GACUGAUUACGCCAGCUUGG (SEQ. ID. NO. 23) or GAC F U F GAU F U F AC F GC F C F AGC F U F U F GG (SEQ. ID. NO. 24)
  • Another aspect of the invention relates to the use of one or more aptamers, or an active fragment thereof, raised against the semi-conserved duffy binding ligand domain 1 ⁇ ,
  • DBL1 ⁇ region of the Plasmodium falciparum erythrocyte membrane protein 1 (PfEMPI) in the treatment of cerebral malaria.
  • PfEMPI Plasmodium falciparum erythrocyte membrane protein 1
  • TAATTATCCCG SEQ. ID. NO. 6
  • a still further aspect of the invention relates to a diagnosis tool for determining the presence of severe malaria, by determining the respsonse of a blood sample to an aptamer raised against the semi-conserved duffy binding ligand domain 1 ⁇ , DBL1 ⁇ , region of the Plasmodium falciparum erythrocyte membrane protein 1 , PfEMPI .
  • a still further aspect of the invention relates to a method for prophylactically and/or therapeutically treating severe malaria by administering to a human being suffering therefrom or risking to suffer therefrom an therapeutically effective amount of an aptamer raised against the semi-conserved duffy binding ligand domain 1 ⁇ , DBL1 ⁇ , region of the Plasmodium falciparum erythrocyte membrane protein 1 , PfEMPI .
  • DBL1 his Protein expression in E. coli.
  • Recombinant DBL1 hls from FCR3S1.2 was expressed as follows, SG13009 (pREP4) cells from Qiagen harboring plasmids pQE-TriSystem His-Sfrep 2 (DBL1 his ), or pQE-60 (DBL1 his ) [22] were grown in LB-medium containing ampicillin (100 ⁇ g/mL) and kanamycin (30 ⁇ g/mL) at either 22°C or 37°C.
  • OD 600 0.8 cells were induced with 0.1 mM IPTG for 3 h; 1 liter cell suspension was harvested (4°C, 25 min, 300Og) and resuspended in 25 ml_ lysis buffer (50 mM NaH 2 PO 4 /NaOH pH 7.4, 300 mM NaCI, 1 mM PMSF (Sigma) 0,05 % Triton x-100, 10 mM imidazole). Cells incubated with Lysozyme on ice for 30 min, and sonicated. Insoluble cell debris was removed by centrifugation (4°C. 30 min, 18.00Og).
  • the supernatant was DNase treated and incubated with 1 ml_ Ni-NTA agarose beads (Qiagen) for 2 h at 4°C with gentle agitation. Beads were pelleted at 100 x g for 3 min. and washed 3 times with 15 mL wash buffer (50 mM NaH 2 PO 4 /NaOH pH 7.4, 300 mM NaCI, 0.05 % Triton x-100, 30 mM Imidazole).
  • wash buffer 50 mM NaH 2 PO 4 /NaOH pH 7.4, 300 mM NaCI, 0.05 % Triton x-100, 30 mM Imidazole.
  • Bound protein was eluted with 400 mM imidazole (Sigma), 50 mM NaH 2 PO 4, 300 mM NaCI which was subsequently removed by dialysis against PBS buffer with 0,05 % Triton X-100 for 18 h at 4°C. Protein purity was estimated by running samples on 10 % SDS-PAGE and finally protein concentration was determined (Bradford 1976). Alternatively, His-tagged DBL1 was purified on FPLC. E. coli extracted soluble fraction was loaded onto Ni-column on FPLC (AmershamBiosciences) with a flow speed of 0.5 mL/min. Bound protein was washed with 5-70 mM Imidazole gradient (60 mL at 1 mL/min). Protein was eluted with 400 mM imidazole and analysed as previously described. All chemicals were obtained from Sigma.
  • RNA molecules were used for the in vitro selection.
  • the library consisted of sequences that had a size of 50 nucleotides flanked by primer binding sites of 20 nucleotides resulting in RNA molecules of 90 nucleotides.
  • T7 RNA transcription was carried out in the presence of 2'F-modified pyrimidine nucleotides to select serum-stable aptamers.
  • the strategy was to enrich for aptamers that would have affinity towards the recombinant DBL1 ⁇ domain from the high rosetting strain FCR3S1.2. Eight rounds of selection and amplification on DBL1 ⁇ pre-bound to nickel beads was performed.
  • RNA/DBL1 ⁇ Hls at 1 :9 generally resulted in high recovery rates, whereas RNA/DBL1 ⁇ His at a ratio of 1 :3 resulted in low levels of recovery (Selected Clones at 12 -25 % recoveries and unselected RNA at 1-4 %).
  • the clones, which showed higher recovery rates were chosen for Electrophoretic Mobility Shift Assays and tested in a live cell assay.
  • RNA sequences of the clones d12, bO2, and eO5, respectively were determined and are represented by the following RNA sequences: d12/1,2 UGCCAACCUUCGAUGCAAGAUAAUACUUUUGAUGGUGUAGUCGUAUUGUU (SEQ. ID. NO. 1)
  • aptamer To improve the half-life time and the stability of the aptamer in blood it can be fluorinated at one or more carbon atoms, preferably all carbon atoms.
  • aptamers disclosed above the following aptamers will then be obtained:
  • RNA/DBL1 ⁇ Hls interaction in solution a number of EMSA experiments were conducted.
  • the protein used for these experiments was freshly eluted from the Ni-NTA beads to avoid long storage and freezing in solution to prevent precipitation of DBL1 ⁇ Hls .
  • the RNA was internally labelled with 32 P-ATP and migration after incubation with DBL1 ⁇ Hls or BSA was analysed on 10 % native polyacrylamide gel.
  • the upper band (177 nucleotide long RNA) seen in figure 4 is an incorrect product of the T7 transcription that is derived from a longer transcription template. This template is a product generated in the previous PCR reaction (binding of PCR primer to en external T7 site in the TOPO-vector).
  • RNA aptamers can disrupt the interaction between DBL1 ⁇ and GAG on the bound uninfected erythrocyte.
  • the aptamers were tested in blood cultures at a concentration of 2 ⁇ g/mL (65 nM) to 12 ⁇ g/mL (387 nM), and with unselected RNA from pool 0 as a negative control. Total disruption was seen at 12 ⁇ g/mL for the clones bO2, d12 and eO5 and a decrease in rosette rate to 35 % in relation to the control was observed at 8 ⁇ g/mL (258 nM) (Figur 7 med rosette status as function of concentration).
  • RNA from pool 0 did not disrupt rosettes in this concentration range (65 - 387 nM), but a small degree of disruption (5-10 % lower than untreated cells) was observed at an unselected RNA concentration of 650 nM to 850 nM.
  • Stability of the 2'F modified aptamers in blood/serum culture was tested. After 3 hours incubation no apparent degradation was observed when running samples on 10 % UREA-PAGE (data not shown). This correlates with previous work done with 2'F modified RNA in blood where the half-life of the RNA was approximately 15 hours.
  • PfEMPI is a key molecule used by P. falciparum to interact with the human host in many ways.
  • the adhesive function of this protein makes it possible for the parasite to adhere to endothelial lining, removing the parasitized erythrocyte from the peripheral circulation and hence avoid spleenic clearance.
  • aptamer can bind its target with extremely high affinity and it has been suggested that they are even more specific than antibodies. Since antibodies must be produced biologically, which would involve expensive cell cultures and eventually animal models, aptamers have an apparent advantage since they can be produced completely in a test tube. Another advantage with the usage of aptamers compared with the use of specific antibodies is in the size difference. Due to their smaller size the RNA aptamers have great potential reaching more buried structures that antibodies are not able to access due to steric hindrance. Additionally the RNA aptamers are easy to chemically modify and elicit little or no immunogenicity in therapeutic applications. Through SELEX technique aptamers have been raised against the semi-conserved DBL1 ⁇ region of PfEMPI .
  • a strategy to minimize the number of groups would have been to increase the selection pressure by decreasing the concentration of RNA and DBL1 ⁇ Hls in later selection rounds, hence discarding RNA aptamers with poor affinity towards the target. The result would be a more homologous pool of RNA binders. Unfortunately the concentration of protein was kept at ⁇ 1 ⁇ M since the quality and solubility of DBL1 ⁇ Hls after elution and dialysis showed to be poor. Another approach to increase affinity and more importantly obtain biological relevant aptamers would be to pan the DBL1 ⁇ Hls selected aptamers on live infected erythrocytes.
  • RNA molecules while adopting a stable duplex hairpin structure with the addition of the negative charge nature of the RNA molecule could theoretically mimic the heparin molecule with its sugar backbone and negative sulphates in place of RNA's phosphate and in the same manner bind to the GAG binding domain of DBL1 ⁇ with a low K D constant.
  • bO2, d12 and eO5 seemed to be stable in regard of binding to DBL1 ⁇ Hls .
  • These aptamers displayed association to the protein when incubated with DBL1 ⁇ Hls at a concentration of 400 nM correlating with the results obtained during the EMSA experiments.
  • the three clones were fluorescein labelled and incubated with live FCR3S1.2 parasites. Fluorescein images were merged with images of the nuclei stained with DAPI. The results show the co- localisation between the fluorescein labelled RNA and the DAPl stained nuclei. The gathered results from this assay show a significant specificity between the selected aptamer clones and the surface of the infected erythrocyte compared to the association between unselected RNA and the infected erythrocyte.
  • An alternative method for a fluorescence based live cell assay would be to end-label the 2'F modified aptamer with biotin thereby maintaining the modification on the UTP and CTP. This greatly enhances stability in blood/serum culture. Exchanging 2'F-d(CU)TP with (CU)TP could also impose a small conformational change in the RNA thereby decreasing the affinity compared to the original 2'F modified RNA. The 2'F atom could have a direct role in binding through interaction with amino acid residues in DBL1 ⁇ . When conducting the live cell assay the rosettes are not manually disrupted prior to the addition of the aptamer.
  • Rosetting could prevent the binding of the labelled aptamers to the DBL1 ⁇ exposed on the surface as the protein is bound and the space occupied by the GAG molecule.
  • the aptamers have a higher affinity towards DBL1 ⁇ than the GAG molecule, they might be able to compete with the binding and thereby exclude and disrupt the GAG/DBL1 ⁇ interaction, visualised as disrupted rosettes. This was tested for the three SELEX selected clones bO2, d12 and eO5. They were all able to disrupt rosettes in an in vitro culture of FCR3S1.2. A concentration as low as 65n M had a limited effect on the rosette rate per se, it did however have an apparent effect when it came to size decrease of the rosettes.
  • a rosette as the indicator of the rosette state in the culture the effect becomes much more apparent.
  • the rosette rate was decreased to approximately 35 % of the control and a concentration of 387 nM was enough to completely disrupt rosettes to single infected erythrocytes with no bound erythrocytes.
  • Unselected RNA did not have any effect at these concentrations but an increase to 650 -850 nM showed a slight effect (5 -10 % disruption). The cause of this effect was never investigated.
  • heparin's ability to disrupt rosettes of FCR3S1.2 a reduction from 100 % to 20 % was observed at a concentration of 100 ⁇ g/mL in the work of vogt et al., which is approximately 7 ⁇ M (Heparin ⁇ 15 kDa).
  • the selected RNA has the ability to disrupt rosettes at 15 times lower concentration than heparin.
  • the clone eO2 was tested for the ability to disrupt rosettes. This clone has a minimal effect on rosette state at 260 nM.
  • the given structures of the aptamers are only theoretical and RNA fingerprinting must be performed to verify the secondary structure of the given RNA.
  • the described results prove that DBL1 specific aptamers have the potential to locate DBL1 on the surface of infected erythrocytes for IFA imaging. More importantly the aptamers could be used as a novel anti - rosetting drug.
  • the next step would be to test the aptamers for rosette disruption and sequestration of parasites in an animal model such as rat to see if the aptamers are active in vivo. Since the field of aptamers as therapeutic drugs is expanding a lot of knowledge on how to increase stability and activity of aptamers in vivo has been obtained. This greatly facilities the success in any future work conducted on aptamers in an in vivo situation. Finally it must be noted that the experimental approach might have a much broader application spectrum, especially in the case of extracellular pathogens where drug delivery through membranes is not required.
  • Bloodstage parasites of P. falciparum strain FCR3S1.2 was cultivated according to standard methods with 10 % AB + Rh + serum added to buffered medium (RPMI supplemented with Hepes, gentamycin and sodium bicarbonate).
  • Recombinant DBL1 ⁇ His from FCR3S1.2 was expressed as follows, SG13009 (pREP4) cells from Qiagen harboring plasmids pQE-TriSystem Hls Strep 2 (DBL1 ⁇ his ), or pQE-60
  • DBL1 ⁇ Hls were grown in LB-medium containing ampicillin (100 ⁇ g/mL) and kanamycin (30 ⁇ g/mL) at either 22 0 C or 37°C.
  • OD 600 0.8 cells were induced with 0.1 mM IPTG for 3 h; 1 liter cell suspension was harvested (4 0 C, 25 min, 300Og) and resuspended in 25 mL lysis buffer (50 mM NaH 2 PO 4 /NaOH pH 7.4, 300 mM NaCI, 1 mM PMSF (Sigma) 0,05 % Triton x-100, 10 mM imidazole).
  • Bound protein was eluted with 400 mM imidazole (Sigma), 50 mM NaH 2 PO 4, 300 mM NaCI which was subsequently removed by dialysis against PBS buffer with 0,05 % Triton X-100 for 18 h at 4°C. Protein purity was estimated by running samples on 10 % SDS-PAGE and finally protein concentration was determined (Bradford 1976). Alternatively, His-tagged DBL1 ⁇ was purified on FPLC. E. coli extracted soluble fraction was loaded onto Ni-column on FPLC (AmershamBiosciences) with a flow speed of 0.5 mL/min. Bound protein was washed with 5-70 mM Imidazole gradient (60 mL at 1 mL/min). Protein was eluted with 400 mM imidazole and analysed as previously described. All chemicals were obtained from Sigma.
  • oligonucleotide B (5'-CGACTGCAGAGCTTGCTACG(N) 5O GGTACCGAGCTCGAATTCCC-S') (SEQ. ID. NO. 19) and oligonucleotide A
  • Oligonucleotides were synthesized and purchased from IBA, Germany. Oligonucleotide B contains a central sequence of 50 randomised nucleotides flanked by constant regions for annealing to oligonucleotide A and a primer site for reverse transcription.
  • Double stranded DNA Library was created by annealing 3 ⁇ M of oligonucleotide A and oligonucleotide B (95°C for 5 min and cool 15 min at 25 0 C), subsequently adding Klenow fragment (Fermentas) in Klenow buffer (Fermentas) at 37 0 C for 2 h.
  • dsDNA is purified by microcon Ym-30 column (Millipore) and eluted in 30 ⁇ l RNAse free water.
  • 2'F-modified library was created by T7 RNA transcription of 40 ⁇ g template using T7 polymerase (Epicentre) in supplied transcription buffer adding DTT (Epicentre) to 10 mM and ATP, GTP, 2'F-dCTP, 2'F-dUTP (Epicentre) to 1.25 mM.
  • RNA was labelled by adding 0.37 MBq [ ⁇ - 32 P]-ATP (Amersham Biosciences) in 20 ⁇ l reaction volume. Reaction ran at 37°C for 5 h and DNA/RNA was precipitated using 0.2 M NaOAc, 70 % EtOH.
  • the first selection cycle was performed with 30 ⁇ g (1 nmol) radioactive labelled 2'F-RNA and a minimum of 60 ⁇ g (1 , 3 nmol) purified DBL1 ⁇ . Subsequent cycles were performed with approx. 300 pmol RNA and varying amounts of protein from 20 ⁇ g to 60 ⁇ g. His- tagged DBL1 ⁇ was bound to Ni-NTA agarose (Qiagen) in cycles 1-4 and 7-8 and to Ni-NTA Magnetic beads (Qiagen) cycles 5 and 6. The concentration of protein and RNA was 800 nM to 1.2 ⁇ M with varying ratios between RNA/Protein (1 :1-4). This concentration range is chosen as other molecules which are suppose to work in a similar manner e.g.
  • heparin sulphate gives the same values in a IOfold higher concentration.
  • a pre-selection on 100 ⁇ l Ni-NTA agarose was performed in every cycle before incubation with DBL1 ⁇ to avoid enrichment of matrix binders.
  • RNA was extracted using phenol/chloroform and precipitated with NaOAc/EtOH. The RNA recovery in each cycle was determined by measuring the radioactivity of all collected fractions during the experiment using a scintillator.
  • RNA concentration was estimated using Nanodrop and ssDNA was generated by adding primer B ( ⁇ '-CGACTGCAGAGCTTGCTACG-S 1 ) in excess and 20 units of M-MuLV-RT (Fermentas) in supplied buffer and 1 mM dNTP. Reaction ran at 37°C for 2 h. 2'F-RNA was partly degraded by addition of 0,1 M NaOH at 37°C for 30 min. ssDNA was purified using Ym-30 microcon column and concentration was determined using Nanodrop. To the ssDNA, oligonucleotide A was added in a 1 :1 ratio. After annealing the two oligonucleotides, full length dsDNA was created by Klenow fill out.
  • primer B ⁇ '-CGACTGCAGAGCTTGCTACG-S 1
  • M-MuLV-RT Fermentas
  • dsDNA was amplified by PCR using Taq DNA polymerase (Fermentas) with a maximum of 14 cycles using primer A ( ⁇ '-GCGTAATACGACTCACTATAG-S 1 ) and primer B. PCR product was pooled and purified and used as template for next SELEX cycle.
  • PCR amplified dsDNA from pool 8 was cloned into TOPO vector pCR ® 4 or pCR ® 2.1 from Invitrogen and transformed into E.coli strain Top10 (Invitrogen).
  • Cells were spread on LB- agar with 100 ⁇ g/mL ampicillin and 30 ⁇ g/mL ⁇ -bromo ⁇ -chloro-S-indolyl-b-D-galacto- pyranoside (X-GaI).
  • White colonies were isolated and insert confirmed by colony-PCR using M13-reverse and -forward primer. Positive clones were streaked onto a 96 well plate and sent to AGOWA (Germany) for sequencing using M13 primer.
  • 70 Sequences were aligned and screened for conserved motifs using the M E ME/MAST system motif discovery search version 3.5.4. (http://meme.sdsc.edu/meme).
  • T7 transcription of 500 ng dsDNA template was done in 20 ⁇ l with 1.25 mM 2'F-dNTP (Epicentre) and NTP (Fermentas), 10 mM DTT (Epicentre), 0.37 Mbq [(X- 32 P]-ATP (Amersham Bioscience ) ran at 37°C for 3 h. Template was digested using 1 unit of DNAse at 37°C for 15 min. RNA was purified using MICROCON ® . RNA concentration was determined using NanoDrop.
  • RNA 25-30 ng internal 32 P-[ ⁇ -ATP] labelled RNA was incubated in a volume of 18 ⁇ l with 4 - 5 ⁇ l (-300 ng) of fresh eluted DBL1 ⁇ His and 3 ⁇ g yeast RNA was added in PBSM.
  • Sample without DBL1 ⁇ H ⁇ s was incubated in the presence of 10 ⁇ g BSA and 4 - 5 ⁇ l 400 mM imidazole elution buffer was added to the BSA sample so the control was identical to the sample incubated with DBL1 ⁇ . Samples were incubated at 37 ° C for 10 min and 6x loading buffer were added (Maniatis).
  • the aptamer RNA was cotranscriptionally labelled with fluorescein.
  • the transcription mix contained NTP labelling mixture (1OmM ATP, 1OmM CTP, 1OmM GTP, 6.5mM UTP and 3.5mM fluorescein-12-UTP, pH 7.5) (Roche Applied Science), 5x T7 transcription buffer, 2 ⁇ l T7 polymerase (50 U/ ⁇ l, Fermentas), sterile RNase free dH 2 O (Fluka), and 200 ng template DNA per 20 ⁇ l reaction. The reaction was performed at 37 0 C for 2 h.
  • the transcription product was DNase treated (New England BioLabs Inc); EtOH/NaOAc precipitated twice and purified using MICROCON ® (MILLIPORE).
  • Negatively charged cells are bound to these slides based on electrostatic adhesion without additional fixation steps allowing the investigation of live late-stage parasitized erythrocytes.
  • the cells were washed with sterile PBS and 500 ng of the RNA sample dissolved in 20 ⁇ l_ PBS 1% BSA was added and allowed to incubate for 30 minutes.
  • aptamer RNA from pool 0 was used as negative control in this experiment. Unbound RNA was removed by washing with sterile PBS and Vectasheild ® Mounting medium with DAPI (4.6-diamidino-2-phenylindole) (Vector) was added. All incubations were carried out at room temperature in a humid chamber. Slides were analyzed with a 100 ⁇ oil immersion lens in a Nikon Optiphot 2 UV microscope.
  • RNA samples for the disruption assay were eluted with water and 1 mM MgCI 2 .
  • Plasmodium falciparum strain FCR3S1.2 was cultured to a parasitimia of 4-5 % in 5 % hematocrit using candle jar technique (ref. Vogt). The cell culture was mixed with the RNA samples to a finale volume of 100 ⁇ l in concentrations from130 nM to 750 nM.
  • Negative controls were performed either with the addition of water/MgCI 2 or unselected RNA generated with T7 transcription from unselected pool 0 dsDNA. Parasites were incubated from 1 to 2Yz hours at room temperature with gentle agitation.
  • Rosetting parasites were defined as infected erythrocytes with more than 2 attached uninfected erythrocytes. Upon auto-agglutination each individual parasite in a cluster was counted as a rosetting parasite. Rosetting rate was calculated as (rosetting parasites/(rosetting parasites + non-rosetting parasites) x 100.
  • Non-labeled RNA was to compete with radioactive 5' labeled RNA for binding to a GST-DBLI ⁇ fusion protein.
  • the recovery of labeled RNA decreases 40 % with the addition of 30 fold molar excess of non- labeled RNA.
  • radioactive labeled d12 was incubated in the presence of 30 fold molar excess of eO2 and pool 0. No decrease was observed with the addition of RNA from these two pools. Furthermore, an additional decrease from 40% to 60% of labeled RNA was demonstrated when 45 fold molar excess of the non-labeled d12 RNA was added.
  • RNA binding to the surface of infected erythrocytes As the selected aptamers were binding to the recombinant DBL1 ⁇ domain the next step was to verify if they could be associated to the surface of the infected erythrocyte. Two of these aptamers was compared to unselected RNA from pool 0 and the non-binding aptamer eO2. Equal molar amounts of RNA were end-labeled with 32 P and incubated with 5 % parasitemia culture of FCR3S1.2. The results demonstrate that the selected RNA is retained 4 fold higher than RNA from unselected pool or the non-binding aptamer eO2.
  • Therapeutic or pharmacological compositions of the present invention will generally comprise an effective amount of the active component(s) of the therapy, dissolved or dispersed in a pharmaceutically acceptable medium.
  • Pharmaceutically acceptable media or carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Supplementary active ingredients can also be incorporated into the therapeutic compositions of the present invention.
  • compositions will be known to those skilled in the art in light of the present disclosure.
  • such compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection; as tablets or other solids for oral administration; as time release capsules; or in any other form currently used, including drops, creams, lotions, salves, inhalants and the like.
  • sterile formulations such as saline-based washes, by surgeons, physicians or health care workers to treat a particular area in the operating field may also be particularly useful.
  • Compositions may also be delivered via microdevice, microparticle or sponge.
  • therapeutics Upon formulation, therapeutics will be administered in a manner compatible with the dosage formulation, and in such amount as is pharmacologically effective.
  • the formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.
  • the quantity of active ingredient and volume of composition to be administered depends on the host animal to be treated. Precise amounts of active compound required for administration depend on the judgment of the practitioner and are peculiar to each individual.
  • a minimal volume of a composition required to disperse the active compounds is typically utilized. Suitable regimes for administration are also variable, but would be typified by initially administering the compound and monitoring the results and then giving further controlled doses at further intervals.
  • the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
  • an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like.
  • suitable binders, lubricants, disintegrating agents and colouring agents can also be incorporated into the mixture.
  • Suitable binders include starch, magnesium aluminium silicate, starch paste, gelatine, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidone, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, polyethylene glycol, waxes and the like.
  • Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, its magnesium or calcium salt and/or polyethyleneglycol and the like.
  • Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthan gum starches, agar, alginic acid or its sodium salt, or effervescent mixtures, and the like.
  • Diluents include, e.g., lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine.
  • the compounds of the invention can also be administered in such oral dosage forms as timed release and sustained release tablets or capsules, pills, powders, granules, elixirs, tinctures, suspensions, syrups and emulsions.
  • compositions are preferably aqueous isotonic solutions or suspensions, and suppositories are advantageously prepared from fatty emulsions or suspensions.
  • the compositions may be sterilized and/or contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, salts for regulating the osmotic pressure and/or buffers. In addition, they may also contain other therapeutically valuable substances.
  • the compositions are prepared according to conventional mixing, granulating or coating methods, respectively, and typically contain about 0.1 to 75%, preferably about 1 to 50%, of the active ingredient.
  • Liquid, particularly injectable compositions can, for example, be prepared by dissolving, dispersing, etc.
  • the active compound is dissolved in or mixed with a pharmaceutically pure solvent such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form the injectable solution or suspension.
  • a pharmaceutically pure solvent such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like.
  • solid forms suitable for dissolving in liquid prior to injection can be formulated.
  • the compounds of the present invention can be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
  • injectables can be prepared in conventional forms, either as liquid solutions or suspensions.
  • preferred compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, inhalants, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
  • the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
  • Other preferred topical preparations include creams, ointments, lotions, aerosol sprays and gels, wherein the concentration of active ingredient would typically range from 0.01% to 15%, w/w orw/v.
  • excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like may be used.
  • the active compound defined above may be also formulated as suppositories using for example, polyalkylene glycols, for example, propylene glycol, as the carrier.
  • suppositories are advantageously prepared from fatty emulsions or suspensions.
  • the compounds of the present invention can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles.
  • Liposomes can be formed from a variety of phospholipids, containing cholesterol, stearylamine or phosphatidylcholines, hi some embodiments, a film of lipid components is hydrated with an aqueous solution of drug to a form lipid layer encapsulating the drug, as described in U.S. Pat. No. 5,262,564.
  • the aptamer molecules described herein can be provided as a complex with a lipophilic compound or non-immunogenic, high molecular weight compound constructed using methods known in the art.
  • An example of nucleic-acid associated complexes is provided in U.S. Patent No. 6,011 ,020.
  • the compounds of the present invention may also be coupled with soluble polymers as targetable drug carriers.
  • soluble polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropyl-methacrylamide-phenol, polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysine substituted with palmitoyl residues.
  • the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and cross- linked or amphipathic block copolymers of hydrogels.
  • the compounds of the invention may also be delivered to the tissue through systemic blood and fluid to the tissues and so is administered by parenteral systemic injection, by intravenous, intramuscular or subcutaneous routes of delivery. Administration via injection of pharmaceutical compositions of the invention may be useful as a supplement to systemic administration of a therapeutic for the treatment of malaria and/or systemic diseases with such manifestations.
  • Compounds of the present invention may also be administered to the tissue in depot or sustained release gel or polymer formulation by surgical implantation of a biodegradable microsize polymer system, e.g., microdevice, microparticle, or sponge, or other slow release transscleral devices, implanted during the treatment of a disease, or by a deliver device, e.g. polymer sustained delivery device.
  • a biodegradable microsize polymer system e.g., microdevice, microparticle, or sponge, or other slow release transscleral devices
  • a deliver device e.g. polymer sustained delivery device.
  • Compounds of the invention may also be administered to the tissue topically, e.g., in drop form loaded with the compound of the invention, or by iontophoresis using electric current to drive drug from the surface to the tissue.
  • the pharmaceutical composition to be administered may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as for example, sodium acetate, and triethanolamine oleate.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as for example, sodium acetate, and triethanolamine oleate.
  • the dosage regimen utilizing the aptamers is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular aptamer or salt thereof employed. An ordinarily skilled physician can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
  • Oral dosages of the aptamer compositions of the present invention when used for the indicated effects, will range between about 0.05 to 7500 mg/day orally.
  • the compositions are preferably provided in the form of scored tablets containing 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100.0, 250.0, 500.0 and 1000.0 mg of active ingredient.
  • Compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily.
  • Infused dosages, intranasal dosages and transdermal dosages of the aptamer compositions of the present invention will range between 0.05 to 7500 mg/day.
  • Subcutaneous, intravenous and intraperineal dosages of the aptamer compositions of the present invention will range between 0.05 to 3800 mg/day.
  • Cerebral dosages of the aptamer compositions of the present invention will range between 0.001 to 10 mg administered, e.g. by injection, from once a week up to once every three months or by sustained release device or formulation.
  • Effective plasma levels of the aptamer compounds of the present invention range from 0.002 mg/mL to 50 mg/mL
  • Effective cerebral levels of the aptamer compounds of the invention can range from 20 nM to 250 ⁇ M.
  • a concentration of 33 nM is enough to provide a disruption capacity, while 380 nM will provide a 100% disruption when it comes to aptamers SEQ. ID. NOs 6 to 9 and corresponding fluorinated ones SEQ. ID. NOs 10 to 12.
  • Figure 2 In vitro selection using random RNA library of sequences with 2'- F substitution on UTP and CTP on purified DBL1 ⁇ bound to either Ni - NTA agarose or Ni - NTA magnetic beads. Selection was done in PBS buffer containing 1mM MgCI 2 with approximately 1 ⁇ M protein and RNA in a 2:1 ration, at 37 0 C and with 30 minutes incubation. Bound RNA was phenol - chloroform extracted and converted to dsDNA by reverse transcription and Klenow fill-in reaction, followed by PCR amplification.
  • FIG. 3 The binding of radioactive labelled RNA on E.coli purified DBL1 ⁇ His . 40 nM 32 P- labeled 2'F-RNA was incubated 45 min at 37 ° C with agitation with 350-400 nM DBL1 ⁇ His in 800 ⁇ l PBSM supplied with 600 ⁇ g yeast RNA. Beads were washed with 2 x 1 ml_ PBS and bound RNA/DBL1 ⁇ His was eluted with 500 mM imidazole. All fractions collected and RNA recovery was estimated by scintillation count.
  • Figure 6 Analysis of live cell assay in fluorescence microscopy, (a) Fluorescein labelled RNA from pool 8 associated to DAPI stained parasitized erythrocytes. The same association of fluorescein labelled RNA is not noticeable towards unparasitized erythrocytes which are seen as black patches, (b) A cut out from the previous picture, (c) Although fluorescein labelled RNA from pool 0 to some extent is associated to DAPI stained parasitized erythrocytes, it is not nearly as distinct as the association between parasite and pool 8 RNA.
  • Figure 7 Preliminary figure of the rosetting disruption effect of clone eO5 and bO2 compared to unselected pool 0 RNA. In the finale figure display concentration of aptamer on x-axis with 1 or 2-4-8-12 ⁇ g/mL and include eO2 as a aptamer with no apparent effect.
  • FIG. 1 Shows status of control and of disrupted rosettes. Rosette disruption of
  • FCR3S1.2 Fluorescence microscopy of cells stained with acridine orange with 4Ox or 2Ox magnification. Images (a) and (c) are control cells which have incubated with 2 ⁇ l water in 98 ⁇ l cells for one hour, (b) and (d) are cells treated with 2 ⁇ l (800 ng) 2 1 F-RNA from SELEX clone d12. In image (c) a giant rosette is enclosed in circle.

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Abstract

La présente invention porte sur un aptamère ou un fragment actif de celui-ci dirigé contre la région semi-conservée du domaine du ligand de liaison Duffy 1α, DBL1α, de la protéine membranaire 1 des érythrocytes de Plasmodium falciparum, PfEMPI, lequel aptamère a un effet à l'encontre de la malaria, en particulier l'accès pernicieux à forme cérébrale sévère.
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SOTIRIS MISSAILIDIS ET AL: "Update: Aptamers as Novel Radiopharmaceuticals: Their Applications and Future Prospects in Diagnosis and Therapy", CANCER BIOTHERAPY & RADIOPHARMACEUTICALS, vol. 22, no. 4, 1 August 2007 (2007-08-01) , pages 453-468, XP55031969, ISSN: 1084-9785, DOI: 10.1089/cbr.2007.357 *

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CN101981188A (zh) 2011-02-23
US9096857B2 (en) 2015-08-04
EP2247728A4 (fr) 2012-08-15
CA2714121C (fr) 2016-03-22
CA2714121A1 (fr) 2009-08-13
US20110003886A1 (en) 2011-01-06
ES2537201T3 (es) 2015-06-03
US9309517B2 (en) 2016-04-12
US20160032293A1 (en) 2016-02-04
US20160040164A1 (en) 2016-02-11
WO2009099378A1 (fr) 2009-08-13
WO2009099378A8 (fr) 2010-12-23

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